Microliter-scale reaction arrays for economical high-throughput experimentation in radiochemistry

The increasing number of positron-emission tomography (PET) tracers being developed to aid drug development and create new diagnostics has led to an increased need for radiosynthesis development and optimization. Current radiosynthesis instruments are designed to produce large-scale clinical batches and are often limited to performing a single synthesis before they must be decontaminated by waiting for radionuclide decay, followed by thorough cleaning or disposal of synthesizer components. Though with some radiosynthesizers it is possible to perform a few sequential radiosyntheses in a day, none allow for parallel radiosyntheses. Throughput of one or a few experiments per day is not well suited for rapid optimization experiments. To combat these limitations, we leverage the advantages of droplet-radiochemistry to create a new platform for high-throughput experimentation in radiochemistry. This system contains an array of 4 heaters, each used to heat a set of 16 reactions on a small chip, enabling 64 parallel reactions for the rapid optimization of conditions in any stage of a multi-step radiosynthesis process. As examples, we study the syntheses of several 18F-labeled radiopharmaceuticals ([18F]Flumazenil, [18F]PBR06, [18F]Fallypride, and [18F]FEPPA), performing > 800 experiments to explore the influence of parameters including base type, base amount, precursor amount, solvent, reaction temperature, and reaction time. The experiments were carried out within only 15 experiment days, and the small volume (~ 10 μL compared to the ~ 1 mL scale of conventional instruments) consumed ~ 100 × less precursor per datapoint. This new method paves the way for more comprehensive optimization studies in radiochemistry and substantially shortening PET tracer development timelines.


Multi-heater platform
The platform was assembled as described in the manuscript from the following components: ceramic heaters (Ultramic CER-1-01-00093, Watlow, St. Louis, MO, USA), , epoxy glue (JB weld, Sulphur Springs, TX, USA), calcium silicate thermal insulation material (McMaster-Carr; Atlanta, GA), 3D-printed nylon piece (Fictiv Inc., San Francisco, CA, USA), DC fans (Sanyo Denki model 9GV3612G301), thermocouple amplifier board (AD8495 Breakout Board, Adafruit, New York, NY, USA), data acquisition module (DAQ; NI USB-6003, National Instruments, Austin, TX, USA), and solid-state relay (SSR, Model 240D05, Sensata-Crydom). Thermal paste (OT-201-2, OMEGA, Norwalk, CT, USA) was used to enhance thermal conductivity between the heaters and the multi-reaction chips. Figure S1 shows a photograph of the entire platform composed of the high-throughput microdroplet apparatus and a separate control box (250 mm x 204 mm x 200 mm) that can be placed outside the shielding or hot cell. CAD models of the platform will be provided upon request. Figure S1. (A) Photograph of fully assembled high-throughput apparatus with control box inside a hot cell.
(B) Photograph with control box located outside the cell to illustrate the small system footprint compared to a conventional radiosynthesizer (ELIXYS, Sofie, Inc., Culver City, CA, USA). C) Electronic wiring diagram of the high-throughput platform.

Thermal simulations
To prevent thermal crosstalk between heaters (i.e., one heater affecting the temperature of a neighboring heater), the heaters were mounted to a frame made of thermally insulating material. Two different types of thermal insulation were explored: Thermo-12 Gold (Johns Manville, Brunswick, GA, USA) and Firetemp -X (Johns Manville). We chose to use Firetemp-X as it could be readily machined, in contrast to Thermo-12 Gold, which we found too flaky and fibrous. To understand if the spacing of the heaters (19.3 mm gap) was enough to prevent thermal crosstalk, thermal modeling was performed on a simplified CAD model of the platform using Solidworks (Dassault Systems, Vélizy-Villacoublay France) with the Solidworks Simulation add-in. The model includes the thermal insulation frame and the heaters. The heaters were modeled as aluminum nitride blocks having a thermal conductivity of 285 W/m-K. The frame was modeled as Firetemp-X; according to manufacturer specifications, the thermal conductivity of this material depends on operating temperature, so we chose a value of 0.094 W/m-K, which corresponds to an estimated operating temperature range of 60-150°C. The model included a thin layer (200 μm) of JB Weld epoxy between the heaters and insulating material, with thermal conductivity of 0.2 W/m-K. A thermal resistivity value of the interface between the heater and the insulation was set to be 0.001 m 2 K/W. This value is derived from both the thermal conductivity of the epoxy and the contact area (allowing for heat transfer) of the heater and insulating material. Bulk ambient temperature and starting temperatures in the model were set to 298 K, and the convective coefficient for stagnant air was modeled as 25 W/m 2 -K. A convection boundary condition using the convective coefficient listed above was applied to all outwardfacing surfaces. Each heater was defined to have a heater power of 150 W. A high-quality mesh was applied to the model resulting in 17239 nodes and 9796 total elements.
A simple steady-state thermal simulation was performed to analyze the overall temperature distribution of the construct with each of the four heaters set to different temperatures. The resulting temperature distribution from the simulation can be seen in Figure S2. The simulation shows that with the designed spacing, heaters are not affected by their neighbors, even for the room temperature (298 K) heater operated adjacent to the hottest (423 K) heater. When probing the insulating material between different pairs of heaters, the highest temperature increase at the midpoint was 10.3 K (between 423 K and 323 K heaters), and the lowest was 3.7 K (between 373 K and 298 K heaters). The temperature change of the insulating material right near the edge of the heater set to 298K was zero confirming adequate insulation of the heater from neighboring heaters The model results were verified empirically on the heating platform through thermal imaging. The heaters on the heating platform were set to different temperatures, and the system was allowed to reach a steady-state. In this study, heaters 1 to 4 (i.e., counterclockwise, starting from the top right) were set to 373K, 323K, 413K, and 305K, respectively. The thermal IR plot can be seen in Figure S3. Positions probed between adjacent heaters on the insulating material show a maximum increase of 5.1 K compared to the temperature of the heater set to the lowest temperature (305 K), suggesting that the insulating material can adequately prevent thermal crosstalk.

Figure S3
: Thermal image of the heater platform. Box 1 through 4 corresponds to areas within heaters 1 through 4. Locations of spot probes between heaters are also shown.
Following the initial steady-state simulation, a transient simulation was performed to estimate the cooling time required for the system to reach room temperature. The model was set up similar to that of the static heating model mentioned above with some minor modifications. The transient model was performed over 600 s with a 1 s step size. Different simulations were performed using different starting temperatures (413 K, 373 K, 323 K) applied to all heaters. The bulk ambient temperature was set to 298 K. To mimic active cooling using fans, a convective coefficient of 200 W/m 2 -K was applied to all surfaces in contact with the cooling airflow. The surfaces are highlighted in Figure S4 by the green cones. All the other exposed surfaces were set with a convective coefficient for stagnant air modeled as 25 W/m 2 -K. A solid mesh was applied consisting of 21594 nodes and 12641 elements.
For each simulation, temperature readings positioned at the center of each heater were measured as a function of cooling time. Due to symmetries in the geometry, all heaters behaved identically. Cooling temperature profiles for heater 1 as a function of different starting temperatures can be seen in Figure  S5. The required cooling time (to 303K) decreases as we decrease the starting temperature. For starting temperatures of 413K, 373K, and 323K, the cooling times were 108 s, 93 s, and 55 s, respectively. Empirical performance (presented in the main paper) was found to have a slower cooling time than the simulation.

Heater calibration and characterization
We initially performed a 2-point linear calibration of thermocouple signal versus temperature by submerging each heater in 2 different water baths (ice water: 0°C; boiling water: 100°C) and measuring the output voltage from the corresponding thermocouple amplifier. Water baths were prepared in 500 mL glass beakers with stir bars, and the temperature was measured independently with a calibrated digital thermometer (53 II B, Fluke, Everett, WA, USA). The temperature stability of the heaters was assessed by setting each heater to a set temperature (50, 100, and 140 °C) and observing the integrated thermocouple measurement for 5 min ( Figure S6). Temperature data were recorded every 0.5 s using the DAQami program (National Instruments) and plotted to examine the heating rate, cooling rate, and temperature stability at the setpoint.

Figure S6
Temperature stability of the four heaters at three different temperatures. In each case, the heater was activated, and once it reached the setpoint, it was maintained at that temperature for 5 min, followed by forced-air cooling. To improve accuracy, a 3-point linear calibration was later performed using a thermal camera (T440-25, FLIR, Wilsonville, OR, USA) to measure the average temperature on the heater surfaces (set at 50, 100, 140 °C, using the original calibration). For each thermal image, the temperature was allowed to stabilize for 5 min before recording the image. The spatial uniformity of temperature distribution was assessed via thermal imaging after this final calibration. Table S1 shows the average thermocouple reading and standard deviation for the plots in Figures S7 and S8. Average and standard deviation were computed from the 5 min region where the temperature had stabilized. All heater temperatures exhibited a standard deviation of <1 °C over time.

Figure S7
Thermal images of all four ceramic heaters (columns) surface at three different temperature setpoints (rows). The color represents the deviation of each pixel from the mean temperature. The dark areas show the pixels that deviate by >2% from the mean. Figure S8. The same data as Figure S7 was replotted in 3D to provide a different illustration of uniformity.
Regions of the heater with deviations >2% were considered to be unusable. Table S2 summarizes the size of unusable regions of each heater at different set temperatures. The maximum width of the unusable region was 1.5 mm across all heaters and temperature setpoints. The maximum fraction of unusable heater surface was 8.3% (always at the edges). Since the maximum width of the unusable region was 1.5 mm, we designed the multi-reaction chips such that the outermost 2.4 mm border was unused, and all reaction sites were entirely located within the usable portion of the heater surface. Figure S9 shows the detailed chip design. Chips are installed onto the heater platform in the orientations shown in Figure S10.

Radio-TLC Methods
In order to use high-throughput radio-TLC for analysis of [ 18 F]Flumazenil, we first investigated different types of TLC plates (normal and RP-18 versions of silica gel 60 F254, Merck KGaA Darmstadt, Germany) and mobile phases from literature 1-4 ( Figure S11). Crude samples were prepared in DMSO:water  Figure 3A). Other parameters in the reaction were chosen to be similar to other syntheses we have adapted to droplet format, i.e., 8 µL reaction volume, 480 nmol of TBAHCO3, and 280 nmol of precursor [5][6][7] . The initial reaction time was chosen to be 2 min, matching the condition reported for a flow microreactor 8 . The crude [ 18 F]Flumazenil product was then collected with 40 μL of 2:1 v/v solvent/water mixture (i.e., the same solvent as used in the reaction). The collection solution loading and collecting were 10 μL at a time and were repeated a total of 4 times to minimize the residue left behind at the reaction. Cerenkov images of chips showing residual activity after collection are shown in Figure 3B of the main paper and radio-TLC data are shown in Figure 3C & D. of the main paper.
Detailed analyses for each individual reaction (collection efficiency, fluorination efficiency, crude RCY, and activity left on-chip) are tabulated in Table S3.  Figure S13A. Chip 1 and 2 explored different base amounts using DMF as the solvent, and chips 3 and 4 explored different base amounts using DMSO as the solvent. All reactions were performed using 280 nmol of precursor in 8 μL of solvent and reacting for 2 min at 200°C. Cerenkov images of chips showing residual activity after collection are shown in Figure S13B and radio-TLC data from reactions is shown in Figure  S14. Detailed analyses for each reaction are tabulated in Table S4.   55.3 ± 4.8 1.9 ± 0.5 1.0 ± 0.2 6.9 ± 1.5 *One reaction was performed incorrectly, and so only n=3 repeats are summarized  Figure S15B and radio-TLC data is shown in Figure S16. Detailed analyses for each individual reaction are tabulated in Table S5.    Figure S17 summarizes the effect of base to precursor ratio on collection efficiency, fluorination efficiency, and crude RCY. This data has all been previously presented above, but here it is reorganized to show the dependence on base to precursor ratio. Table S6 tabulates the values of base to precursor ratio used as the x-axis in Figure S17.

Reaction time and solvent
The study of the effect of reaction time on the synthesis of [ 18 F]Flumazenil was conducted as shown in Figure S18A.  Figure S18B and radio-TLC data are shown in Figure S19. Detailed analyses for each individual reaction are tabulated in Table S7.     Figure S20B, and radio-TLC data is shown in Figure 21. Detailed analyses for each individual reaction are tabulated in Table S8.   We then explored the effect of temperature using NMP as reaction solvent for the radiosynthesis of  Figure  S22B, and radio-TLC data are shown in Figure S23. Detailed analyses for each individual reaction are tabulated in Table S9, and the results are plotted in Figure S24. The optimal temperature was 200 °C, giving a crude RCY of 19.1 ± 0.6% (n=4).     Figure S25A. After drying, the subsequent fluorinations were performed with 280 nmol of precursor in 8 µL of DMF, DMSO, or NMP and reacted at 200°C for 0.5 min. Cerenkov image of the chip showing residual activity after collection is shown in Figure S25B, and radio-TLC data is shown in Figure S26. Detailed analyses for each individual reaction are tabulated in Table S10.    19.1 ± 0.6 (n=4) ------

RCY (decay-corrected; %)
11.6 (n=1) Δ N.R. 9.0 ± 1.0 (n=6)* 8 26 ± 4 15-20 Δ 30 Δ ‡ The value corresponding to the optimized condition is not clearly specified, so the whole range reported in the paper is indicated # Not reported, but amount of KHCO3 was computed based on the amount of precursor and an indicated 1.9:1 molar ratio of base to precursor. The amount of K222 was in turn computed based on the reported 1.1:1 molar ratio of K222 to KHCO3. N.R. = Not Reported * Calculated from shorter and higher-yield SPE purification method instead of HPLC Δ Isolated yield (i.e., not formulated) § 20 min for radiosynthesis and HPLC purification plus an estimated ~15 min additional time for formulation 10 6 Optimization of [ 18 F]PBR06 synthesis 6

.1 Precursor amount and solvent
Effect of precursor amount experiments were conducted as depicted in Figure S27A.  Figure S27B, and radio-TLC data is shown in Figure S28. Detailed analyses for each individual reaction are tabulated in Table S12.    Figure S29A.
In the subsequent fluorination, chips 1 and 2 used thexyl alcohol:MeCN (1:1 v/v) mixture as a reaction solvent and chips 3 and 4 used DMSO. All reactions used 160 nmol of precursor and were performed at 100°C for 5 min. Cerenkov images of chips showing residual activity after collection are shown in Figure  S29B, and radio-TLC data from reactions is shown in Figure S30. Detailed analyses for each individual reaction are tabulated in Table S13.

Reaction temperature and solvent
The experimental design for exploring temperature and solvent effect on the radiosynthesis of [ 18 F]PBR06 was described in Figure S31A Figure S31B, and radio-TLC data are shown in Figure S32. Detailed analyses for each individual reaction (collection efficiency, fluorination efficiency, crude RCY, and activity left on chip) are tabulated in Table S14.

Reaction time and solvent
The study of the effect of reaction time on the synthesis of [ 18 F]PBR06 was conducted as shown in Figure  S33A.  Figure S32B and radio-TLC data are shown in Figure S34. Detailed analyses for each individual reaction are tabulated in Table S15.

Base type and solvent
Finally, we explored the use of different type of base / phase transfer catalyst, comparing Kryptofix (K222) and K2CO3 versus TBAHCO3 as shown in Figure S35A.  Figure S35B, and radio-TLC data is shown in Figure S36. Detailed analyses for each individual reaction are tabulated in Table S16.   , followed by fluorination with 160 nmol of precursor in 8 µL of solvent for 0.5 min at different temperatures. To conserve chips, temperatures were explored sequentially, using 4 fresh reaction sites each time. (Two chips were needed in total.) Radio-TLC data are shown in Figure S37. Detailed analyses for each individual reaction are tabulated in Table S16 and the results are plotted in Figure S38. We observed that the temperature could be lowered to 90 °C without compromising performance. Table S17 compares our approach with other literature macroscale reports.    Figure S39B, and graphical representation is shown in Figure S41A in the main paper. Detailed analyses for each individual reaction are tabulated in Table S18.

Precursor concentration and reaction time
Another study of the impact of precursor concentration and reaction time was conducted as depicted in Figure S40A.  Figure S40B.
Detailed analyses for each individual reaction are tabulated in Table S20, and the results are plotted in Figure S41B.    Figure S42B, and radio-TLC data is shown in Figure S43.
Detailed analyses for each individual reaction are tabulated in Table S21.      (Figure S45A). A subsequent injection was performed to confirm purity (Figure S45B), and a co-injection with Flumazenil reference standard was performed to confirm product identity ( Figure S45C). The crude product showed minimal UV impurities.

[ 18 F]PBR06
Crude [ 18 F]PBR06 was injected in HPLC to isolate the product (Figure S46A). A subsequent injection was performed to confirm purity (Figure S46B), and a co-injection with PBR06 reference standard was performed to confirm product identity ( Figure S46C). The crude product showed minimal UV impurities.

[ 18 F]Fallypride
Crude [ 18 F]Fallypride was injected in HPLC to isolate the product (Figure S47A). A subsequent injection was performed to confirm purity (Figure S47B), and a co-injection with Fallypride reference standard was performed to confirm product identity ( Figure S47C). The crude product showed minimal UV impurities.

[ 18 F]FEPPA
Crude [ 18 F]FEPPA was injected in HPLC to isolate the product (Figure S48A). A subsequent injection was performed to confirm purity (Figure S48B), and a co-injection with FEPPA reference standard was performed to confirm product identity ( Figure S48C). The crude product showed minimal UV impurities. 10 Clinical-scale radiosynthesis Summaries of high activity droplet syntheses of [ 18 F]PBR06 carried out with different starting activity are shown in Table S23 and Figure S49.